Evaluation of Various Escherichia coli Strains for Enhanced Lycopene Production

Lycopene is a carotenoid widely used as a food and feed supplement due to its antioxidant, anti-inflammatory, and anti-cancer functions. Various metabolic engineering strategies have been implemented for high lycopene production in Escherichia coli, and for this purpose it was essential to select and develop an E. coli strain with the highest potency. In this study, we evaluated 16 E. coli strains to determine the best lycopene production host by introducing a lycopene biosynthetic pathway (crtE, crtB, and crtI genes cloned from Deinococcus wulumuqiensis R12 and dxs, dxr, ispA, and idi genes cloned from E. coli). The 16 lycopene strain titers diverged from 0 to 0.141 g/l, with MG1655 demonstrating the highest titer (0.141 g/l), while the SURE and W strains expressed the lowest (0 g/l) in an LB medium. When a 2 × YTg medium replaced the MG1655 culture medium, the titer further escalated to 1.595 g/l. These results substantiate that strain selection is vital in metabolic engineering, and further, that MG1655 is a potent host for producing lycopene and other carotenoids with the same lycopene biosynthetic pathway.


Table 1. Plasmids and oligonucleotides within this study.
Plasmids Description pA-SRAI E. coli dxs, dxr, ispA, and idi genes under P BAD promoter control were cloned in a plasmid of p15A origin and a chloramphenicol resistance gene pWA-EBI, EIB, BEI, BIE, IEB, IBE D. wulumuqiensis R12-derived enzyme genes (crtE (E), crtB (B), crtI (I)) were cloned in various orders and transcribed under a synthetic promoter (BBa_J23118) control. The plasmid containing the three genes harbored ColE1 origin and an ampicillin resistance gene.

HPLC Lycopene Measurement
A single metabolically engineered strain colony was incubated overnight in 5 ml of LB at 37°C and 230 rpm to measure lycopene production. Cells were inoculated into 50 ml of LB with 1% L-arabinose (Bio Basic, CAS#5328-37-0, Canada) and appropriate antibiotics, followed by incubation at 30°C and 200 rpm for 60 h. All experiments were performed in the dark because lycopene is light-sensitive, and lycopene measurements were repeated thrice. A Biotek Synergy H1 plate reader (Winooski, VT, USA) measured cell growth (OD 600 ). At 48 and 60 h postincubation, cells from 50 ml of culture broth were harvested through centrifugation at 7600 ×g and 4°C for 5 min. Cell pellets were washed once with distilled water, resuspended in acetone, and incubated at 55°C for 15 min. After centrifugation (7,600 ×g, 25°C, 10 min), HPLC was used to analyze the supernatant for lycopene quantification.
For lycopene analysis, 20 μl of a sample was analyzed by isocratic HPLC with a ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 5 μm; Agilent, USA) and a mobile phase composed of 80% acetone, 15% methanol, and 5% isopropanol at a constant flow rate of 1 ml/min for 20 min at 30°C. A commercially available lycopene (Sigma-Aldrich, USA) was used as a standard, and acetone-extracted lycopene was detected at 472 nm. All experiments were conducted under dark conditions to avoid lycopene isomerization by light [34,35]. Fig. 1 illustrates the metabolic pathway toward lycopene. E. coli can inherently produce the lycopene precursor farnesyl diphosphate (FPP); however, it lacks the enzymes that convert FPP to lycopene. First, three D. wulumuqiensis R12-derived genes (crtE, crtB, and crtI genes) were cloned into a mid-copy plasmid containing a ColE origin (pWA plasmid) and transcribed by a synthetic constitutive promoter (BBa_J23118) for lycopene biosynthesis (Fig. 2). As the lycopene pathway consumes the intermediate metabolites in the glycolysis pathway, a mid-strength synthetic promoter (BBa_J23118) was employed to avoid cell growth diminution. Second, to drive more metabolic fluxes from the glycolysis pathway to FPP, the lycopene biosynthetic pathway precursor, four E. coli genes (dxs, dxr, ispA, and idi) were cloned into a mid-copy plasmid (pA-SRAI) and overexpressed under P BAD promoter control. Like the lycopene pathway genes, a relatively weak promoter prevented growth defects.

Polycistronic crtE, crtB, and crtI Gene Clusters on E. coli DH5α Lycopene Production
Six polycistronic gene clusters of the three genes were constructed and evaluated in E. coli DH5α with or without the plasmid pA-SRAI to investigate the lycopene biosynthetic gene (crtE, crtB, and crtI) order effect on lycopene titer ( Fig. 2A). The six polycistronic gene clusters (pWA-EBI, pWA-EIB, pWA-BEI, pWA-BIE, pWA-IEB, and pWA-IBE; Table 1) were separately introduced into E. coli DH5α and the transformed cells were cultured in an LB medium for 48 h, as previously described [24]. The lycopene titer ranged from 0 to 1.0 mg/l without pA-SRAI internal flux enhancement (Fig. 2B). When the introduced pA-SRAI plasmid enhanced the internal metabolic flux towards FPP, the lycopene titer escalated to 24.1 mg/l (pWA-IEB). Since the crtI-crtE-crtB polycistronic gene cluster exhibited the highest lycopene titer with pA-SRAI co-expression, this gene cluster was selected for strain screening.

Evaluating 16 E. coli Strains for Lycopene Production
Even strains of the same species may express eclectic production capabilities [29,36,37]. Thus, selecting the best strain is a critical step in metabolic engineering. Therefore, a selected polycistronic gene cluster (pWA-IEB) and internal flux-enhancing (pA-SRAI) plasmids were co-transformed into 16 E. coli strains to identify the best strain for lycopene production: 13 K-12 strains (DH5α, SURE, MG1655, JM110, XL10-Gold, XL1-Blue, LS5218, W3110, W3110ΔlacI, SM-10, TOP10, JM109, and NEB Turbo), one B strain (BL21(DE3)), one K-12 and B hybrid strains (HB101), and one W strain. Table 2 conveys the 16 E. coli strain genotypes. The transformed cells were cultured in an LB medium for 48 h. As we aimed to screen diverse strains in this study, no genomic engineering was conducted in the strains chosen. In addition, since the improper use of defined media could excessively reduce the lycopene titer past distinguishable strain capacities, we used LB and 2xYT as the high-nutrient media. Fig. 3A depicts the lycopene titers of the 16 strains. The MG1655 strain (141 mg/L) expressed the highest lycopene titer. Thus, the best strain for lycopene production was E. coli MG1655 with the crtI-crtE-crtB gene cluster and dxs, dxr, ispA, and idi gene expressions. The other five gene clusters were also evaluated to confirm whether the selected polycistronic gene cluster was the best in MG1655 (Fig. 3B). Consistent with the DH5α gene cluster evaluation results, the crtI-crtE-crtB cluster achieved the highest lycopene titer in MG1655. Currently, the most common E. coli strains for lycopene production are W3110, DH5α, XL1-Blue, and Bl21(DE3) [24,26,28]. However, these strains demonstrated a notably inferior lycopene production capability by comparison with MG1655, suggesting that the employment of MG1655 along with preexisting metabolic engineering strategies could substantially increase lycopene production.

Lycopene Production Increase from a 2 × YTg Growth Enhancement Medium
The metabolically engineered MG1655 strain reached a stationary phase at 12 h (Fig. 4A). We cultured cells in 2 × YT and 2 × YTg media to investigate if growth enhancement could further increase the lycopene titer. Glycerol is a viable carbon source for β-carotene and lycopene production [36,[38][39][40][41][42], as it increases glyceraldehyde 3phosphate and pyruvate, which are imperative intermediates in central carbon metabolism extension to the MEP pathway [43,44]. We determined that 2 × YTg significantly increased lycopene production due to the observed cell growth increase (Figs. 4A and 4B) [36,42]. Interestingly, no significant lycopene titer or growth increase in LB, LB (+glycerol), and 2 × YT indicated that enriched nutrients and glycerol significantly increase cell growth and lycopene production. Furthermore, glycerol decreased cell growth rate during the initial phase, while the rich media (LB and 2 × YT) revealed an increase. However, cells incubated in the rich media reached a stationary phase sooner than cells grown with glycerol. This finding is potentially due to glycerol altering metabolism [45,46], although this theory requires future study.

Discussion
Selecting an optimal base strain is the first crucial step in metabolic engineering, as it could increase the lycopene titer from 0 mg/l (SURE) to 141 mg/l (MG1655). In this study we observed a substantial variety of lycopene titers in the 16 E. coli strains, even though they are derived from the same species. Consequently, the final lycopene titer achieved from the optimal E. coli strain MG1655, gene cluster, and medium combination was 1,595 mg/l, while the worst strains with non-optimal gene clusters did not express a detectable lycopene titer. To our knowledge, this titer was superior to the previous highest lycopene titer obtained from E. coli (1,240 mg/l) in a flask culture [17,47], which emphasizes the value of strain selection. These results confirm that base strain selection is vital for enhanced substance production, and E. coli MG1655 is the optimal strain for lycopene metabolic engineering.